PROJECT SUMMARY We are combining two innovative technologies, NovAb's Variable Lymphocyte Receptor (VLR) monoclonal antibody technology and NatGlycan's Oxidative Release of Natural Glycans (ORNG) technology, to create a high throughput, high capacity process for discovery of a new class of binding reagents for biologically relevant glycans, i.e., the glycans of tissues, cells and glycoproteins. Such reagents are essential to detecting, localizing, quantifying, and establishing biological function(s) of specific glycans in normal and disease cells and tissues, and may additionally lead to development of new diagnostic and therapeutic modalities. The absence of such reagents is considered a significant barrier to understanding the role of glycans in health and disease. VLRs are the antigen receptors of the adaptive immune system of jawless vertebrates, lamprey and hagfish, are composed of tandemly arranged highly diverse leucine-rich repeat (LRR) structural motifs with diversity comparable to the human immunoglobulin (Ig) antibody repertoire, and possess a distinctive more rigid ?-sheet binding site geometry vs Ig antibodies that is entirely contained within a single polypeptide structure. Vertebrates split into jawless and jawed vertebrate lineages approximately 550 million years ago. The evolutionary separation of jawless vertebrates from humans and other mammals, and the distinctive binding site geometry of VLRs potentially provide novel specificities not accessed with Ig antibodies. This expectation is supported by the discovery of VLRs that bind glycans with affinity and specificity superior to that typically achieved with Ig antibodies and indicate that VLRs are uniquely well- suited to binding and discriminating glycan structures. ORNG is a technology for releasing glycans from natural sources, e.g. tissues, cells and glycoproteins, purifying the released glycans, determining structures of the purified glycans, and chemically modifying glycans for attachment to solid surfaces. In combining these technologies biological sources, e.g., human cells or glycoproteins, are utilized to immunize lamprey and these same biological sources utilized to derive glycans via NatGlycan's ORNG technology with which to select/screen for lamprey VLR monoclonal antibodies that specifically bind each of the purified glycans. We expect this process to yield dozens of monoclonal VLRs, each with a distinct glycan binding specificity from each lamprey immunization regimen with cellular and glycoprotein immunogens. The VLR-ORNG technology is therefore potentially a transformative for the rapid, high throughput production of specific binding reagents for the diverse glycan structures that comprise the human glycome